The broad objective of the proposed research project entails the development of an integrated liquid phase-based platform for addressing and facilitating the inherently challenging qualitative and quantitative measurements involved in glycoproteomics. The proposed platform will allow the accurate in-depth sub- glycoproteomics, e.g., aberrantly fucosylated and sialylated glycoproteins, which are indicative of cancers. Thus far, the traditional approach for glycoproteomics involves many discontinuous steps each of which may introduce measurement discrepancies that are significantly compounded throughout the overall process. This has prohibited the accurate realization of glycoproteomics profiling that are pertinent to a given investigation. Thus, it is the aim of this proposal to provide remedies to these shortcomings. Toward this end, the following two major specific aims will be pursued: (i) Develop, characterize and optimize a multistage platform to function in "cascade" whereby the information of interest, e.g., specific glycoproteomics, will be extracted, captured, concentrated and fractionated using various modes of chromatography including immunoaffinity, protein A affinity, protein G4 affinity, lectin affinity chromatography and reversed phase chromatography. In the cascade, the information of interest is transferred continuously form the first stage to the last stage, thus eliminating discontinuous sample manipulation which causes sample loss and propagation of experimental biases from step to step. The platform allows the extracted serum glycoproteins to be transferred from column to column (i.e., from one chromatographic mode to another) while staying in the liquid phase. (ii) Demonstrate the uniqueness and effectiveness of the proposed platform in the accurate and reproducible differential (i.e., comparative) quantitative analysis of sub-glycoproteomics by LC-MS/MS. We will target fucosylated glycoproteins and sialylated glycoproteins in the aim that the two sub-glycoproteomes are implicated in the onset of several types of cancer (i.e., the two sub-glycoproteomes carry the signature of a diseased state). In fact, increases in sialylation and fucosylation of glycan structure have been linked to cancer progression. The success in differential sub-glycoproteomics measurements between healthy and diseased states will certainly have significant clinical diagnostic potential. The two specific aims constitute a series of closely related projects, which if funded will effectively contribute to advancing glycoproteomics, providing liquid phase-based platform of potential use in the clinical and biomedical research and training undergraduate and graduate students in bio-separations and biomedical processes. PUBLIC HEALTH RELEVANCE: We propose to develop a liquid phase-based platform to target serum sub-glycoproteomics, e.g., fucosylated and sialylated glycoproteins. The two sub-glycoproteomes are associated with diseases, and therefore their isolation and profiling is of biomedical significance. The platform will operate in "cascade" assembling various modes of chromatography to accomplish the removal of nuisance high abundance proteins, the capturing of sialylated and fucosylated low abundance glycoproteins, and their subsequent fractionation and quantitation by coupled liquid chromatography - mass spectrometry (LC-MS/MS).